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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 04 March to 01 May 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with relevant guideline and under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Manston Road, Margate, Kent
- Weight at study initiation: At the start of the treatment the males weighted 289 to 355 g and the females weighted 195 to 238 g.
- Housing: Upon arrival, the animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays.
- Diet (e.g. ad libitum): A ground diet 5002 was offered ad libitum.
- Water (e.g. ad libitum): The animals were allowed free access to water. Main water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ℃
- Humidity (%): (55 ± 15)%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: 04 March 2003 To: 01 May 2003

No additional data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared as direct dietary admixture. For each dose level, an appropriate aliquot of test substance was weighted and then added to approximately one third of the required amount of untreated diet in a mixing bowl. An initial mix of the test material and diet was performed using a Hobart QE200 mixer for ten minutes. Further untreated diet was then added to the initial mix to give the required concentration and the entire amount was then mixed for ten minutes using the Hobart H800 mixer.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): untreated diet

No additional data
Details on mating procedure:
- M/F ratio per cage: one male to one female
- Length of cohabitation: basis for a period to eighteen days
- Proof of pregnancy: The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating.
- After successful mating each pregnant female was caged (how): Mated females were then separated from the male and housed individually during the period of gestation and lactation.

No additional data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the start of the study, samples of the test diet preparation were analysed for achieved concentration, homogeneity and stability of test material in diet. The results of analysis of the original preparation showed the test substance to be stable for at least fourteen days.
Duration of treatment / exposure:
The test substance was administered throughout maturation (dosed for two weeks prior to pairing), mating, gestation and up to Day 5 of lactation.
Frequency of treatment:
Once daily
Details on study schedule:
None stated
Remarks:
Doses / Concentrations:
0, 1000, 5000, 20000 ppm
Basis:
nominal in diet
Initial dose level
Remarks:
Doses / Concentrations:
0, 900, 4500, 18000 ppm
Basis:
nominal in diet
Adjusted dose level
No. of animals per sex per dose:
Ten animals per sex per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of a dietary range-finding study.

No additional data
Positive control:
None stated
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the normal working week and once daily at weekend/bank holiday.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: During the maturation and mating period the parental generation animals were weighted weekly. Following mating the parental males were weighted weekly until termination. Parental generation females showing evidence of mating were weighted on Day 0, 7, 14 and 20 post coitum. Parental generation females with a liver litter were weighted on Day 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. During the maturation period, food consumption was recorded for each cage of adults weekly. For females showing evidence of mating, food consumption was recorded for the periods covering Days 1 to 7, 7 to 14 and 14 to 20 post coitum. For females with live litters, food consumption was recorded for the period covering Days 1 to 4 post partum.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OTHER:
Functional Observations
Prior to the start of treatment and on Days 8, 15 and 22, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and five selected females per group on Day 22 for males and Day 4 of lactation for females, together with an assessment of sensory reactivity to difference stimuli.

Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group on Study Day 13 (prior to mating). Blood samples were obtained from the lateral tail vein. Animals were not fasted prior to sampling.

Pregnancy and Parturition: Each pregnant female was observed at 0830, 1230 and 1630 hours at or around the period of expected parturition. At weekends, observations were carried out at 0830 and 1230 hours only. The following was recorded for each females:1) Data of mating; 2) Data and time of observed start of parturition; 3) Data and time of observed completion parturition.
Oestrous cyclicity (parental animals):
None stated
Sperm parameters (parental animals):
None stated
Litter observations:
At the observation of completion of parturition, the number of live and dead offspring was recorded.

The following observations were recorded for all individual offspring alive on the particular day of observation.
Bodyweight: Individual offspring weights were recorded on Day 1 and 4 post partum.
Offspring Numbers and sex: The number of offspring was recorded daily, up to weaning, and reported for Day 1 and 4 post partum.
Clinical signs: The clinical condition of individual offspring was observed daily and any findings recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at Day 5 of lactation
- Maternal animals: All surviving animals at Day 5 of lactation

GROSS NECROPSY
- Gross necropsy consisted of both internal and external abnormalities.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues listed next were examined from five males and five females selected from the control and high dose groups. Histopathology was extended to examination of the spleen for five males and five females selected from both the low and intermediate dose groups.
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone and bone marrow (femur including stifle joint), Pituitary, Bone and bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Rectum, Caecum, Salivary glands (Submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Thyroid/parathyroid, Eyes, Trachea, Gross Lesions, Vagina, Heart, Coagulating gland, Ileum, Skin (hind limb), Jejunum, Spinal cord (cervical), Kidneys, Spleen, Liver, Stomach, Lungs (with bronchi), Testes, Lymph nodes (cervical and mesenteric), Thymus, Mammary gland, Urinary bladder, Muscle (Skeletal), Uterus, Oesophagus.
The following list of tissues were examined from the remaining unselected males and females from the control and high dose groups: Epididymides, Seminal vesicles with coagulating gland, Ovaries, Uterus and cervix, Pituitary, Vagina, Prostate, Testes.
The following list of organs were weighted for all adult males and females at necropsy where applicable: Adrenals, Liver, Brain, Ovaries, Epididymides, Spleen, Heart, Testes, Kidney and Thymus, paired organs were weighted together.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at Day 5 of age.
- These animals were subjected to postmortem examinations macroscopically.

GROSS NECROPSY
- Gross necropsy consisted of internal and external abnormalities.

No additional data
Statistics:
The following parameters were analysed statistically, where appropriate using the test methods as follows:
Adult male and female bodyweight during the maturation, gestation and lactation periods, adult male food consumption, female food consumption during maturation, gestation and lactation, litter size, litter weight, individual offspring bodyweight offspring landmarks of physical development, haematology, blood chemistry and organ weights.
Values were analysed to establish homogeneity of group variance using Levene’s test followed by one-way analysis of variance. If the variances were unequal subsequent comparisons between control and treated groups were performed using a pairwise “T” test Comparison method. If variances were equal subsequent comparisons between control and treated groups were performed using Dummett’s Multiple Comparison Method. For haematology and blood chemistry values an additional regression analysis of all values was also performed.

Adult pre-coital intervals, female gestation lengths, offspring reflexological responses and litter sex ratio, relative organ weights. Individual values were compared using the Kruskal-Wallis non-parametric rank sum test. Where significant differences were seen, pairwise comparison of control values against treated group values was performed using performed using Mann-Whitney “U” test.

Histopathology: For those tissues from selected animals only.
Chi-squared analysis for differences in the incidence lesions occurring with an overall frequency of 1 or greater.
Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed conditions.
Probability values were calculated as follows: P<0.001, +++, ---***; p<0.01, ++, --**; p<0.05, +, -*; p<0.1, (+), (-)(*); p≥0.1, NS, (not significant), Where plus signs indicate positive differences from the control group, and minus signs negative differences. Asterisks refer to overall between group variation.
Reproductive indices:
For each group the following were calculated:
Mating Index (%)= (Number of animals mated)/(Number of animals paired) × 100;
Pregnancy Index (%) = (Number of pregnant females)/(Number of animals mated) × 100
Offspring viability indices:
Viability Index (%) = (Number of pups alive on Day 4)/(Number of pups alive on Day 1) × 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no mortalities during the course of the study. There were no clinical signs of toxicity throughout the course of the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
At 20000 ppm, there were inconsistent differences in bodyweight gain for males throughout, and females prior to pairing, compared to controls. Female bodyweight gain during gestation was lower than controls resulting in significant bodyweight differences during the final week of gestation and early lactation. There were no significant effects upon food consumption except for a statistically significant difference in female food consumption during the final week of gestation.
At 5000 ppm, a similar pattern of reduction in bodyweight gain during gestation for females was observed but no significant effect upon female food consumption.
At 1000 ppm, there were no significant differences in female bodyweight gain and food consumption during the study period.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The results showed that the highest dose level of 20000 ppm achieved a test substance intake excess of 1000 mg/kg/day for males and females and this was maintained even after the reduction to 1000 ppm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no significant treatment-related effects upon reproductive performance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
An increase in absolute and relative liver weight for females only when compared to control at 20000 ppm. An increase in liver weight for females only in 5000 ppm.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related macroscopic post mortem findings for adult males or females.

HISTOPATHOLOGY (PARENTAL ANIMALS)
At 20000 ppm, histopathology showed a reduction in the severity grades for splenic extramedullary haemopoiesis for both males and females compared to controls but this is of questionable toxicological relevance; At 5000 nppm and 1000 ppm, histopathology showed lower severity grades of splenic extramedullary haemopoiesis for male only.

OTHER FINDINGS (PARENTAL ANIMALS)
At 20000 ppm, Laboratory investigation showed a significant increase in both Activated Partial Thromboplastin Time and Clotting time for male only when compared to control values, There were no significant blood chemistry changes. There were no significant effects on blood chemistry or haematology at 5000 and 1000 ppm.
Dose descriptor:
NOAEL
Effect level:
61 mg/kg diet
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
> 1 225 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive performance
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
VIABILITY (OFFSPRING)
There were no significant treatment-related effects upon offspring from birth to Day 4 post partum.

CLINICAL SIGNS (OFFSPRING)
There were no significant treatment-related effects upon offspring clinical condition.

BODY WEIGHT (OFFSPRING)
There were no significant treatment-related effects upon offspring bodyweight gain from birth to Day 4 post partum.

GROSS PATHOLOGY (OFFSPRING)
There were no significant treatment-related macroscopic post mortem findings for offspring that died from birth to Day 4 post partum. At scheduled termination of offspring, there were no significant treatment-related findings.

OTHER FINDINGS (OFFSPRING)
Offspring Sex Ratio: There were no significant treatment-related effects upon intra and inter litter sex ratios.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 225 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Offspring viability
Reproductive effects observed:
not specified
Conclusions:
At dose levels of 5000 ppm and above there was evidence of treatment-related effects upon the adult. At a dose level of 1000 ppm and above the only significant findings was lower grades of severity of splenic extramedullary haemopoiesis which is considered not to be an adverse finding. No evidence of effects upon reproductive performance or subsequent offspring viability in utero and early lactation as seen. Offspring development was comparable to control values.
The “No Observed Adverse Effect Level” for effects upon adults was 1000 ppm and for reproductive performance and offspring viability was in excess of 20000 ppm.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1 (reliable without restriction)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

According to an OECD 422 study, conducted to GLP, the NOAEL (rats) for reproductive performance and offspring viability of Methylenebis (dibutyldithiocarbamate), is greater than 20,000 ppm (1,225 mg/kg diet) (Safepharm Laboratories Limited, 2006).


Short description of key information:
The NOAEL (rats) for reproductive performance and offspring viability of Methylenebis (dibutyldithiocarbamate), is greater than 20,000 ppm (1,225 mg/kg diet) (Safepharm Laboratories Limited, 2006).

Justification for selection of Effect on fertility via oral route:
This study was carried out with rat based on OECD 422.

Effects on developmental toxicity

Description of key information
Methylenebis (dibutyldithiocarbamate)  has an acute (oral) LD50 of >1,600 mg/kg, a chronic (oral) LOAEL of 5,000ppm (320 mg/kg diet), a reproductive toxicity LOAEL of 20,000 ppm (1,225 mg/kg diet) and is not expected to be absorbed via the oral route in accordance with Lipinski's rule of five. In addition, exposure of Methylenebis (dibutyldithiocarbamate) to humans via skin contact is considered minimal due to appropriate Risk Management Measures (RMM) identified in the Chemical Safety Report, this endpoint is waived.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

According to Column 2 of Annex IX of the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) legislation, developmental toxicity does not need to be conducted if the substance is of low toxicological activity, it can be proven that no systemic absorption and human exposure is unlikely. Methylenebis (dibutyldithiocarbamate) has an acute (oral) LD50 of >1,600 mg/kg, a chronic (oral) LOAEL of 5,000ppm (320 mg/kg diet), a reproductive toxicity LOAEL of 20,000 ppm (1,225 mg/kg diet) and is not expected to be absorbed via the oral route in accordance with Lipinski's rule of five. In addition, exposure of Methylenebis (dibutyldithiocarbamate) to humans via skin contact is considered minimal due to appropriate Risk Management Measures (RMM) identified in the Chemical Safety Report.

Therefore, based upon low toxicological activity and no systemic absorption, this endpoint is waived.

Justification for classification or non-classification

Reproductive toxicity:

Based on a NOAEL of greater than 20,000ppm (1,225 mg/kg diet), in accordance with Regulation No 1272/2008, Methylene bis (dibutyldithiocarbamate) is not classified for reproductive toxicity.